Cytokines such as tumor necrosis factor (TNFalpha),interleukin-1 (IL- 1beta), and interferon-gamma (IFNgamma) have profound effects on islet cells: 1) they inhibit insulin synthesis and secretion by the beta-cell, 2) they destroy beta-cells, and 3) they are produced by immune and inflammatory cells infiltrating islets in the insulitis of insulin- dependent diabetes mellitus (IDDM), and the course of immune rejection of transplanted islets. The rationale for these studies is that the identification of the molecular mechanisms mediating cytokine-induced beta-cell cytotoxicity will facilitate the development of novel preventive and therapeutic strategies. the initial signal transduction pathways involved have not been fully characterized, although,as recently demonstrated in other cells, some of the actions of cytokines may involve stimulation of the sphingomyelinase and phospholipase A2 pathways. Our Preliminary Data have identified the sphingomyelinase/ceramide signal transduction pathway as an important component of the effects of IL-1beta and TNFalpha on islet cells, and we have developed the following hypothesis: the cytotoxic effects of TNFalpha, IL-1beta and INFgamma on the beta-cell are due, in part, to activation of the sphingomyelinase and phospholipase A2 signal transduction pathways. In order to test this hypothesis, we propose the following Specific Aims. Aim 1 is to determine the role of the sphingomyelinase/ceramide signal transduction pathway in mediating the effects of TNFalpha,IFNgamma, and IL-1beta on the beta-cell by biochemically quantitating the substrates, enzymatic activity, and end-products of this pathway. Membrane-permeable short-chain ceramide analogues will be used to reproduce the cytotoxic effects of IL-1beta, IFNgamma,and TNFalpha on the beta-cell. Aim 2 is to determine which beta-cell phospholipase A2 isoform (type II 14 kDa secretory isoform, 85 kDa cytosolic enzyme, Ca2+-independent activity) is induced and regulated by IL-1beta, IFNgamma, and TNFalpha using molecular (RT-PCR), immunological (Western blotting), and biochemical assays. Aim 3 is to elucidate the mechanisms whereby TNFalpha, IFNgamma, and IL- 1beta affect specifically the beta-cell of the endocrine pancreas. By using purified alpha and beta cells, pure beta-cell lines, and islets devoid of antigen-presenting cells, we will localize to specific cell types the components of the target signal transduction pathways. Aim 4 is to develop strategies to prevent cytokine-induced beta-cell death using chemical inhibition of phospholipase A2, RNA antisense transfection approaches, and antisense DNA infection of islets targeted against phospholipase A2. Aim 5 is to determine the role of the sphingomyelinase and phospholipase A2 signal transduction pathways in the pathogenesis of inflammatory lesions of pancreatic islets using in vivo models of insulitis and immune rejection, which are characterized by elevated cytokine levels.